Comparison of the gut microbiome of sacbrood virus-resistant and -susceptible Apis cerana from South Korea

Honey bees are important pollinators for the conservation of the ecosystem and agricultural products and provide a variety of products important for human use, such as honey, pollen, and royal jelly. Sacbrood disease (SD) is a devastating viral disease in Apis cerana; an effective preventive measure for SD is urgently needed. In this study, the relationship between the gut microbiome of honey bees and SD was investigated by pyrosequencing. Results revealed that sacbrood virus (SBV)-resistant A. cerana strains harbour a unique acetic acid bacterium, Bombella intestini, and the lactic acid bacteria (LAB) Lactobacillus (unclassified)_uc, Bifidobacterium longum, B. catenulatum, Lactococcus lactis, and Leuconostoc mesenteroides in larvae and Hafnia alvei, B. indicum, and the LAB L. mellifer and Lactobacillus HM215046_s in adult bees. Changes in the gut microbiome due to SBV infection resulted in loss of bacteria that could affect host nutrients and inhibit honey bee pathogens, such as Gilliamella JFON_s, Gilliamella_uc, Pseudomonas putida, and L. kunkeei in A. cerana larvae and Frischella_uc, Pantoea agglomerans, Snodgrassella_uc, and B. asteroides in adult bees. These findings provide important information for the selection of probiotics for A. cerana larvae and adults to prevent pathogenic infections and keep honey bees healthy.


Gut microbiome of SBV-resistant A. cerana.
The gut microbiota of SBV-resistant A. cerana larvae of R and H strains belonged to only two phyla, Proteobacteria and Firmicutes. The gut microbiota of SBV-resistant larvae showed a lower diversity than that of SBV-susceptible larvae. The difference of gut microbiota was significant between SBV-resistant H strain and SBV-susceptible strain (p = 0.0001). The major species in the R strain were G. apicola and Lactobacillus_uc, and only Bombella intestini was identified in the H strain ( Fig. 2; Table S1).
The gut microbiota prevalent in SBV-resistant A. cerana adults in R and H strains comprised the same phyla, namely Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. Further, gut microbiota of SBV-resistant strains was not significantly different from that of susceptible strains, p = 0.6839 and 0.5862 between susceptible strains and resistant R strain and between susceptible strains and resistant H strain, respectively. In comparison with the gut microbiome of SBV-susceptible strains, SBV-resistant strains showed three new species, H. alvei, L. mellifer, and Lactobacillus HM215046_s, and lacked Frischella_uc and P. agglomerans. In addition, Gilliamella JFON_s (17.01%) and Gilliamella_uc (17.66%) were more abundant in the resistant H strain than in the resistant R strain exhibiting Gilliamella JFON_s and Gilliamella_uc at 6.45% and 8.48%, respectively, and those in the susceptible strains were 6.37% and 7.53%, respectively. G. apicola was dominantly detected in the susceptible strain (18.35%). However, it was detected at only 9.98% and 8.71% in R-and H-resistant strain, respectively ( Fig. 2; Table S1).

Lactic acid bacteria in A. cerana.
The LAB species L. kunkeei (19.29%) was predominant in the gut of SBV-susceptible A. cerana larvae, while B. asteroides (8.08%), L. mellis (5.35%), and L. kimbladii (3.27%) were identified in the gut of SBV-susceptible adults. There was a major loss of LAB in the gut of SBV infected larvae; only one LAB species, L. kimbladii (0.022%), was detected in the diseased larvae. Similarly, in diseased adults, Figure 1. Gut microbiome of SBV-susceptible Apis cerana. Bacterial species were identified from the gut of sacbrood virus (SBV)-susceptible A. cerana adults and larvae from both healthy (−) and sacbrood disease (SD)affected (+) honey bees collected from Cheongju province. ("uc" indicates unclassified species; "JFON_s" and "JFZW_s" indicate uncultured species of genera Gilliamella and Snodgrassella, respectively).  Table S2). Although the number of LAB in the larvae of SBV-resistant A. cerana was lower than that in susceptible larvae, the difference was not remarkable. B. longum was present in both R and H strains. LAB identified in larvae of only the R strain were L. apis, L. mellifer, L. melliventris, Lactococcus lactis, and Leuconostoc mesenteroides, and LAB in larvae of only the H strain comprised B. catenulatum. There was no notable difference between the SBVsusceptible and -resistant adults in terms of species number and abundance of LAB ( Fig. 3; Table S2). The gut microbiota of adults and larvae of two artificially bred A. cerana strains, R (individual immunity) and H (social immunity), was identified and confirmed to be capable to resist SBV ("uc" indicates unclassified species; "JFON_s" and "JFZW_s" indicate uncultured species of genera Gilliamella and Snodgrassella, respectively; HM215046_s indicates the NCBI accession number of Lactobacillus species). The p-value of diversity index between the larvae of SBV-resistant R strain and susceptible strain was 0.0059 ( Fig. 4a), and between resistant H strain and susceptible strain was 0.091 (Fig. 4b). Meanwhile, the p-value of comparison between resistant adult and susceptible adult was 0.006 (Fig. 4c). Furthermore, the gut bacterial community in larvae of SBV-resistant strain and susceptible strain (Fig. 5a,b), and in adult bee of the two strains ( Fig. 5c) can be distinguished using non-metric multidimensional scaling (NMDS) based on Bray-Curtis indices. The NMDS stress value of resistant larvae strain R and H compare to susceptible strain was 0.0978 and 0.0979, respectively, and the value of adult bee comparison was 0.1059. Alpha diversity and NMDS of other comparisons were shown in Figs. S1, S2, and S3.

Discussion
In this study, we found differences in the microbiota of SBV-susceptible and -resistant A. cerana in Korea for the first time. The microbiota of SBV-resistant A. cerana larvae included Bombella intestini and Lactobacillus_uc, which were absent in SBV-susceptible A. cerana larvae. Members of the core gut community in honey bees have been known to be S. alvi (class: Betaproteobacteria; family: Neisseriales), G. apicola, F. perrara (Gammaproteobacteria; Orbales), Alphaproteobacteria, and Lactobacillae (Firmicutes; Lactobacillaceae) 19 . Acinetobacter sp.,  www.nature.com/scientificreports/ Fructobacillus fructosus, Commensalibacter intestine, and L. kimbladii have been identified in the gut microbiome of A. mellifera in a previous study [19][20][21][22] . Thus, Bombella intestini was a species unique to SBV-resistant A. cerana, when compared with A. mellifera and SBV-susceptible A. cerana. Bombella intestini is an endosymbiotic acetic acid bacterium found in bumble bees (Bombus bimaculatus) 23,24 , which was also identified in A. cerana for the first time in this study. SBV has been known as a gut-affinity virus, leading to damage of the gut of larvae and subsequent rotting of the diseased larvae 6 . One of the preventive measures for honey bee diseases is probiotics, which provide nutrients, protect the attachment of pathogens to cell surfaces, and create an acidic environment that is harsh for the survival of pathogens 20,[25][26][27] . Therefore, probiotics could have positive effects on the survival of SBV-infected A. cerana. The unique microbiota in SBV-resistant adults in comparison with the SBV-susceptible adults included H. alvei and the LAB L. mellifer, Lactobacillus HM215046_s, and B. indicum. H. alvei was assumed to be an opportunistic pathogen of honey bees 28 . However, the function of this bacterium in SBV-resistant adult bees remains unclear. Besides, LAB unique to the larvae of SBV-resistant A. cerana compared to those of susceptible strains were B. catenulatum, B. longum, L. apis, L. mellifer, L. melliventris, Lactococcus lactis, and Leuconostoc mesenteroides. LAB produce organic acids, known as anti-microbial metabolites, inhibiting the growth of spoilage and pathogenic microorganisms 29 . Therefore, microbiota unique to SBV-resistant A. cerana in each developmental stage could be useful for the development of probiotics for disease prevention in honey bee larvae and adults.
LAB play important roles in the production and preservation of honey bee nutrients 30 . In addition, several studies have showed that LAB was helpful in increasing the size of honey bee colony by increasing the egg-laying capacity of the queen 31,32 and resistance to honey bee diseases such as nosemosis 31,33,34 and varroosis 31 . Common LAB in all the larvae and adults of A. cerana in this study were B. asteroides, L. helsingborgensis, L. kimbladii and L. mellis, of which L. helsingborgensis and L. kimbladii were also isolated from A. mellifera and described for the first time in 2014 22 . They showed the ability to produce acid from d-glucose, d-fructose, d-mannose, N-Acetylglucosamine, arbutin, salicin, and d-tagatose 22 . However, further studies on the usefulness of the LAB species unique to SBV-resistant A. cerana might be important to understand whether they provide practical resistance against SBV infection.
Comparison of the gut microbiota of healthy and SD-affected larvae in SBV-susceptible A. cerana revealed that SD progression resulted in the loss of Gilliamella JFON_s, Gilliamella_uc, Pseudomonas putida, and L. kunkeei. Some of these bacterial species have been identified to have important functions in the gut of honey bees. For instance, Gilliamella spp. are endosymbionts and play a role in degrading polysaccharides that could affect the absorption of host nutrients 35 , Pseudomonas putida has the ability to degrade neonicotinoid insecticides 36 , and L. kunkeei is known to inhibit opportunistic pathogens 37 . Furthermore, SBV infection in adult bees resulted in the loss of Frischella_uc, P. agglomerans, Snodgrassella_uc, B. longum, L. kullabergensis, and L. kunkeei and a decrease in the abundance of B. asteroides and L. mellis in the gut. Frischella spp., such as F. perrara, is known to stimulate the immune system of A. mellifera 38 , and P. agglomerans acts as a biocontrol agent against fire blight and human facultative pathogens 39 . G. apicola, L. kimbladii and B. asteroides produce acidic products via fermentation 40,41 . G. apicola was also demonstrated to have the ability to break down various carbohydrates that are potentially toxic to honey bees 42 . A. mensalis, Snodgrassella spp., and F. perrara were found to stimulate the immune system of A. mellifera 38 . However, the mutualistic interaction between the host and the Apibacter spp. remained unclear 43,44 . Other LAB species are capable of digesting polysaccharides and producing bioactive compounds that possess the potential to act as antimicrobials 35,40,45 . Therefore, supplements of these bacteria as probiotics could be helpful to maintain a healthy gut environment and provide efficient protection against SD.
There was no common essential microbiota in the larvae. However, in adults, the common microbiota between susceptible and resistant strains were seen; these included G. apicola, Gilliamella JFON_s, Gilliamella_uc, Snodgrassella JFZW_s, L. kimbladii, L. mellis, A. mensalis, and B. asteroids. The common microbiota in adults are vital for survival 46 . Evidence suggests that differences in the gut microbiota could have originated from differences in the natural environment and the queen lineages of honey bees [46][47][48][49][50] . The comparison of the gut microbiome from healthy, susceptible adult honey bees collected from two provinces, Jeju and Cheongju, showed that the major bacterial species present in the gut of adults were not notably different (p = 0.493). However, the diversity of the gut microbiome in adults was higher in Cheongju than in Jeju (Fig. S4a). Interestingly, considerable differences were observed in the gut microbiome of larvae collected from the two provinces (p = 0.0436; Fig. S4b). There were also differences observed in the gut microbiome collected in different seasons. The identification of the gut microbiome collected in June and October in Cheongju province was not significantly different, p = 0.5802 and 0.2039 for adult and larvae, respectively. The results revealed that P. agglomerans was predominant in adults in October, while G. apicola was predominant in June (Fig. S5a). In case of the larval gut, L. kunkeei, Gilliamella_uc, and Snodgrassella_uc were found in October and Gilliamella_uc and G. apicola in June (Fig.  S5b). Therefore, further studies are required to understand the influence of environmental or natural factors, such as type of pollen and nectar in sampling sites, on the actions of gut bacteria in honey bees and interaction between the host and the bacteria.
In conclusion, the gut microbiota unique to SBV-resistant A. cerana was identified. This study revealed that the SBV infection resulted in the loss of gut microbiota that could affect host nutrients and inhibit certain opportunistic pathogens in A. cerana. The results of this study can provide important information for designing and developing developmental stage-and strain-based probiotics that could be formulated including the essential common bacterial group, specific species in resistant strains, and the important common LAB group. The probiotics could be important to protect A. cerana from pathogenic infections, and for further research on preventing severe SD outbreaks and economic losses to apiaries. . SD colonies were identified by clinical signs, which were the irregular capping of combs, the pulled-out larvae out of hives, shrunk or rotten larvae, and larvae inclined to the cell wall. The presence of SBV in larvae and adult bees was confirmed by SBV detection by real-time PCR using specific primers (forward primer: 5′-AGA AGT TTT GGT GTA TAT GCG AGG-3′ and reverse primer: 5′-CTG CGC AGT TTC ATC TTC ATC TTC-3′, and probe 5′-HEX-AAA TAG ACC AAG AAG GGA ATC AGA TAA TCC-BHQ-1-3′) 10 .

Collection of samples.
Larvae and adults of SBV-susceptible and -resistant A. cerana were collected and transported to the laboratory in refrigerated conditions (4 °C). Guts were isolated from both adults and larvae and stored in Eppendorf tubes at − 20 °C before sending them for microbiome analysis by pyrosequencing. Number of larvae and adults collected from each colony varied from one to three depending on the quality of extracted gut and sequencing result. Information of collected samples is shown in Table 1.
Extraction of nucleic acid. The collected gut samples were added to Lysing Matrix E tubes containing ceramic beads (MP Biochemicals GmbH, Eschwege, Germany). After adding PBS (400 μl) to the samples, they were homogenised with a Precellys 24 Tissue Homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). Nucleic acid was extracted using the FastDNA Spin Kit for Soil (MP Biochemicals GmbH, Eschwege, Germany) following the manufacturer's instructions.
PCR amplification and illumina sequencing. PCR amplification was performed using primers targeting V3 to V4 regions of the 16S rRNA gene using the extracted DNA. Primers of 341F (5′-TCG TCG GCA GCG TC-AGA TGT GTA TAA GAG ACA G-CCT ACG GGNGGC WGC AG-3′) and 805R (5′-GTC TCG TGG GCT CGG-AGA TGT GTA TAA GAG ACA G-GAC TAC HVGGG TAT CTA ATC C-3′) were used for amplification of the target gene. Amplification was carried out under the following conditions: initial denaturation at 95 °C for 3 min, followed by 25 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final elongation at 72 °C for 5 min. Next, secondary amplification for attaching the Illumina NexTera barcode was performed using the i5 forward primer (5′-AAT GAT ACG GCG ACC ACC GAG ATC TACAC-XXXXXXXX-TCG TCG GCA GCG TC-3′; X indicates the barcode region) and i7 reverse primer (5′-CAA GCA GAA GAC GGC ATA CGA GAT -XXXXXXXX-AGT CTC GTG GGC TCGG-3′). The conditions for secondary amplification were identical to those for the first one, except that the amplification cycle was set to 8.
The PCR product was confirmed by using 2% agarose gel electrophoresis and visualised under a Gel Doc system (BioRad, Hercules, CA, USA). The amplified products were purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). Equal concentrations of purified products were pooled together and short fragments (non-target products) were removed using the Ampure beads kit (Agencourt Bioscience, Beverly, MA, USA). The quality and product size were assessed using a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. Mixed amplicons were pooled and sequencing was carried out at ChunLab, Inc. (Seoul, Korea), using the Illumina MiSeq Sequencing system (Illumina, San Diego, CA, USA) according to the manufacturer's instruction. www.nature.com/scientificreports/ MiSeq pipeline method. Processing of raw reads was conducted via a quality check (QC) and filtering of low quality (< Q 25) reads by Trimmomatic 0.32 51 . After the QC pass, paired-end sequence data were merged together using PandaSeq 52 . Primers were then trimmed with ChunLab's in-house program at a similarity cut off of 0.8. Sequences were denoised using Mothur's 53 pre-clustering program, which merged sequences and extracted unique sequences allowing up to 2 differences between sequences. The EzTaxon database was used for taxonomic assignment using BLAST 2.2.22 and pairwise alignment was used to calculate similarity 54,55 . Uchime and the non-chimeric 16S rRNA database from EzTaxon were used to detect chimeras on reads that had a best hit similarity rate of less than 97% 56 . Sequence data were then clustered using CD-Hit and UCLUST, and alpha diversity analysis was carried out 57 . It is to be noted that in this database, the uncultured phylotype is tentatively given the hierarchal name assigned to the DDBJ/ENA/GenBank accession number with the following suffixes: "_s" (for species), "_g" (genus), "_f " (family), "_p" (phylum) 58 .
Diversity analysis. Alpha diversity with Simpson index was used to compare gut microbial diversities between group of collected honey bee, and the p value was calculated with Wilcoxon t-test. The difference of gut bacterial community between SBV-resistant and -susceptible honey bee strain was also determined by NMDS distancing by Bray-Curtis index. The analysis was done by using Vegan community ecology package version 2.5-7, and visualized by using ggplot2 package version 3.3.5.
Statistical analysis. Comparison of microbiota in larvae with SD to larvae without SD, in adult with SD to adult without SD, in SBV-resistant larvae to SBV-susceptible larvae, in resistant adult to susceptible adult, and the abundance of different microbiotic strains in different seasons and different regions was done using Mann-Whitney U tests (non-parametric) from program PAST version 4.03. The differences between samples were considered to be significant when p < 0.05.

Data availability
All data generated or analysed during this study are included in this published article and its Supplementary Information files.